(i) Field of the Invention
The present invention relates to the use of new antigens identified in the parasite Trichinella in the context of the diagnosis and the prevention of trichinellosis.
(ii) Description of the Related Art
Trichinellosis is a zoonosis associated with the consumption of meat infested with the parasite Trichinella (MURRELL et al., 2000).
This nematode of the class Adenophorea belongs to the family Trichinellidae which comprises 8 species and 3 genotypes that are related, in 2 phylogenetically distinct groups: on the one hand, the encapsulated trichinae (T. spiralis; T. nativa; T. britovi; T. murrelli; T. nelsoni) which infest mammals, end on the other hand, the nonencapsulated trichinae (T. pseudospiralis; T. papuae; T. zimbabwensis) which infest mammals, birds and reptiles (GASSER et al., 2004). All these species can infest humans.
The biological cycle of the parasite is autoheteroxenous: it takes place entirely in the same host, which is successively the definitive host (carrying adult parasites) and the intermediate host (carrying infesting larvae) (BOIREAU et al., 2002). The passing of the infesting larvae from host to another is necessary in order for a new cycle to be carried out. This passage occurs through the ingestion of raw or barely cooked meat contaminated with the larvae. During digestion, the latter are released, and penetrate the intestinal epithelium, where they will mature into sexual adult (Ad) worms. The fertilized females subsequently expel newborn L1 larvae (L1NN) which reach the striated muscles via the lymphatic circulation and the blood stream. These L1NN larvae penetrate the muscle cells (infesting development stage L1M: L1 muscle larva), of which they bring about the dedifferentiation into feeder cells surrounded by a protective collagen capsule which is thick in the case of encapsulated trichinae, very thin in the case of “nonencapsulated” trichinae.
Although trichinellosis is asymptomatic in animals, human infestation is reflected, during the initial intestinal phase, by diarrhea associated with nausea, vomiting and violent abdominal pain, while the symptoms associated with the muscle invasion phase are characterized by the combination of fever, facial edema and myalgia (CAPO & DESPOMMIER, 1996). Ocular, pulmonary, gastrointestinal, cardiac and neurological attacks can also add to this clinical picture of trichinellosis, the progression of which can be lethal. The chronic nature of the infestation, marked by persistent muscle pain in patients, is associated with the survival of the parasite in the feeder cell.
The specific treatment of human trichinellosis with anthelminthics is all the more effective if the diagnosis of the infestation is made early so as to allow action against all the parasitic stages and especially before the formation of the protective collagen capsule around the L1M larvae (FOURESTIE et al., 1988).
The epidemiological data have demonstrated a geographical distribution of the parasite in all parts of the world, associated with a method of transmission involving many species of the wild-type fauna which also maintain a domestic infestation cycle mainly represented by the pigs (DUPOUY-CAMET, 2000).
Epidemics of human trichinellosis, an emerging or reemerging zoonosis, constitute a real public health problem throughout the world owing to dietary habits and hygiene controls that are not always effective (MURRELL & POZIO, 2000). These epidemics essentially involve pig and wild boar meat and also horse meat (BOIREAU et al., 2000).
The prevention of human contamination therefore involves cooking meat right through and improving rearing conditions and/or conditions for controlling animal trichinellosis (pigs, horses, wild boar and other wild animal species sensitive to Trichinella) (BOIREAU et al., 2002).
The screening techniques for trichinellosis can be divided into two categories: 1) direct detection of the L1M larvae, by trichinoscopy (microscopic observation of a meat fragment), or after artificial digestion of muscle samples, and 2) indirect detection by various immunological methods, for detecting antibodies directed against the Trichinella antigens.
Each of the developmental stages of the parasite: adult (Ad), newborn larva (L1NN) and muscle larva (L1M) has a corresponding specific antigen profile.
It is antigen preparations derived from L1M-stage larvae which are currently used for immunodiagnosis. This is because the antigen fractions of the two early stages Ad and L1NN are difficult to purify, and it had not been possible to identify immunodominant antigens associated with one and/or the other of these two stages up until now.
Either preparations of total soluble antigen, obtained by lysis of the larvae, centrifugation of the lysate and recovery of the supernatant, or, more commonly, excretion/secretion antigens (E/S antigens) are principally used.
The excretion-secretion antigens are produced when L1M larvae are placed under survival conditions in a culture medium; they originate from a particular organ, called the stichosome, which comprises about fifty discoid cells, the stichocytes. The stichocytes contain granules, the content of which is evacuated by a canaliculus into the lumen of the parasite's esophagus. This content, which is very highly antigenic, constitutes a part of the excretion-secretion antigens. These antigens form a complex mixture of proteins, containing in particular a group of glycoproteins (called TSL1 antigens) bearing a specific carbohydrate molecule, known only in Trichinella and present in all the species of this parasite, beta-tyvelose.
The preparations of excretion-secretion antigens which are currently used as a reference in terms of immunodiagnosis of trichinellosis are obtained from culture medium of Trichinella spiralis L1M larvae. After culture for 18 to 20 hours, the medium is recovered by filtration and then concentrated (GAMBLE et al., 1983; GAMBLE et al., 1988).
The principal drawback of the preparations of total soluble antigen is their lack of specificity. Antigen cross reactions with other parasitoses are commonly observed. The excretion-secretion antigens make it possible to obtain a better specificity. However, in both cases, it is difficult to produce standardized batches of antigen in large amounts.
The saccharide structure containing beta-tyvelose, which represents an immunodominant epitope of E/S antigen preparations (REASON et al., 1994; U.S. Pat. Nos. 5,541,075 and 5,707,817), has been synthesized chemically, and its use for the immunodiagnosis of Trichinella has been proposed.
This reagent has good specificity, but its sensitivity appears to be lower than that of E/S antigen preparations. In addition, the chemical synthesis of this structure remains expensive and laborious to carry out.
Another problem encountered in the context of the serological diagnosis of Trichinella is the existence of a “blind window” of detection corresponding to the early stages of infestation, which is reflected by false-negative results. In addition, in horses, gradual disappearance of the antibodies has been observed 25 weeks after infestation.
The inventors have undertaken to identify immunodominant antigens associated with the early stages of Trichinella infestation and which can be used for the serological diagnosis of trichinellosis, in order to provide means for obtaining early, specific and sensitive detection of Trichinella infestations, both in humans and in animals. With this aim, they have investigated whether there existed, among the products of the genes expressed by Trichinella at the L1NN stage and/or at the Ad stage, proteins which would possess the desired antigenic properties.
In this context, they have discovered that a Trichinella spiralis protein which is part of the proteins expressed specifically at the L1NN stage in this organism, constitutes an immunodominant antigen, allowing early detection of the humoral response directed against Trichinella, and that it is also conserved between various species of Trichinella. 
This protein will hereinafter be referred to as NBL1. The complete cDNA sequence encoding this protein and also the polypeptide sequence which is deduced therefrom are respectively accessible on Genbank under numbers AF331160 and AAK16520 (also known as Swissprot Q9BJL7); these sequences are annotated as “serine protease SS2, specific for newborn larvae”. These sequences are also respectively reproduced in the attached sequence listing under numbers SEQ ID NO: 1 and SEQ ID NO: 2. A partial cDNA sequence encoding this protein and also the polypeptide sequence which is deduced therefrom are respectively accessible on Genbank under numbers AY491941 and AAR36900 (also known as Swissprot Q6RUJ3).
The inventors have also shown that the immunoreactivity associated with the humoral response directed against NBL1 is located in the C-terminal part of this protein, and have identified an immunodominant epitope responsible for this reactivity.
Furthermore, the inventors have identified, using a cDNA library of mixed early Ad+L1NN stages of T. spiralis, a new gene, hereinafter referred to as 411.
The sequence of this gene is represented in the attached sequence listing under the number SEQ ID NO: 3, and that of its translation product under the number SEQ ID NO: 4. This product of translation of this gene is related (78.7% identity) to an E/S antigen, known as Tp21-3 protein, identified in T. pseudospiralis (AAF79206; NAGANO et al., 2001), and also to the product of translation of the hypothetical ORF 17.20 of T. spiralis (AAB48489), with which it exhibits 86.6% identity.
The 411 gene translation product also makes it possible to detect, at an early stage, the humoral response directed against various species of Trichinella. 
In addition, each of the NBL1 and 411 antigens has made it possible, in the assays carried out, to detect animals infested with Trichinella which were not detected with the other antigen, nor with the E/S antigen, at least during the period D15-D30 post-infestation (pi).
The combination of the NBL1 antigen (or of an immunodominant epitope thereof) with the 411 antigen therefore makes it possible to improve the sensitivity of the diagnosis, in particular at the early stages of infestation (15 to 20 days after infestation).
Consequently, a subject of the present invention is the use of an antigenic polypeptide recognized by anti-Trichinella antibodies as a reagent for detecting anti-Trichinella antibodies in a biological sample, characterized in that said polypeptide is chosen from:
a) a polypeptide comprising an immunodominant epitope of the NBL1 antigen, which epitope is defined by the sequence PSSGSRPTYP (SEQ ID NO: 5);
b) a polypeptide, also referred to hereinafter as 411 antigen, comprising amino acids 25-175 of the sequence SEQ ID NO: 4 (which represent the mature form of the 411 protein), or comprising a sequence having at least 80%, and by order of increasing preference, at least 85%, 90% or 95% identity with the sequence of amino acids 25-175 of the sequence SEQ ID NO: 4.
A subject of the present invention is more particularlv a method for detecting the presence of anti-Trichinella antibodies in a biological sample, which method is characterized in that it comprises:                bringing said biological sample into contact with one or more polypeptide(s) a) and/or one or more polypeptide(s) b), as defined above, under conditions which allow the formation of an antigen/antibody complex with the anti-Trichinella antibodies possibly present in said sample;        detecting, by any appropriate means, the antigen/antibody complex possibly formed.        
Generally, said biological sample is a serum sample. It can be obtained from any individual (mammals, bird or reptile) belonging to a species that can be infested with Trichinella, and in which it is desired to detect the presence of this parasite. Advantageously, it is a sample obtained from a mammal, for example from a farm animal, or from a human patient.
Advantageously, a mixture comprising one or more polypeptide(s) a) and/or one or more polypeptide(s) b) as defined above is used.
This combination makes it possible in particular to broaden the spectrum of reactivity, relative to each of the polypeptides used individually.
The polypeptides a) defined above, with the exclusion of the whole NBL1 antigen identified by Genbank accession number AAK16520, and of its fragment identified by Genbank accession number AAR36900, are also part, as such, of the subject of the present invention.
Among these polypeptides in accordance with the invention, mention will in particular be made of the polypeptides containing one or more of the following sequences: the sequence: PSSGSRPTYPSSGSR (SEQ ID NO: 6); the sequence PSSGSRPTYPYTGSR (SEQ ID NO: 7); the sequence RPTSPSSGSRPTYPS (SEQ ID NO: 8).
This encompasses, for example, fragments of the C-terminal region of the NBL1 antigen: mention will in particular be made of the fragments containing the following sequence:
(SEQ ID NO: 11)ENSPEGTVKWASKEDSPVDLSTASRPTNPYTGSRPTSPSSGSRPTYPSSG SRPTSPSSGSRPTYPSSGSRPTYPSSGSRPTYPYTGSRPTPQKPVFPSYQ KYPPAVQKYIDSLPSGTQGTLEYTVTQNGVTTTT,which corresponds to amino acids 326-459 of the sequence SEQ ID NO: 2; and subfragments of this sequence SEQ ID NO: 11, in particular those comprising the following sequence:
(SEQ ID NO: 9)PSSGSRPTYPSSGSRPTSPSSGSRPTYPSSGSRPTYPSSGSRPTYP,which corresponds to amino acids 363-409 of the sequence SEQ ID NO: 2, and more particularly, those comprising the following sequence:
(SEQ ID NO: 10)SRPTNPYTGSRPTSPSSGSRPTYPSSGSRPTSPSSGSRPTYPSSGSRPTY PSSGSRPTYPYTGSRPT,which corresponds to amino acids 349-415 of the sequence SEQ ID NO: 2.
The polypeptides b) defined above, with the exception of those identified by GenBank accession numbers AAF79206 and AAB48489, are part, as such, of the subject of the present invention. Preferred polypeptides are in particular the polypeptide of sequence SEQ ID NO: 4, or the polypeptide corresponding to amino acids 25-175 of the sequence SEQ ID NO: 4, and also the polypeptides having at least 90%, or preferably at least 95%, identity with the sequence SEQ ID NO: 4, or with the sequence of amino acids 25-175 of the sequence SEQ ID NO: 4.
The present invention encompasses in particular chimeric polypeptides comprising one or more copies of the sequence PSSGSRPTYP (SEQ ID NO: 5), or of a fragment of the NBL1 antigen containing this sequence, and/or one or more copies of a polypeptide b) as defined above, optionally fused to one or more other heterologous sequence(s).
A subject of the present invention is also the polynucleotides encoding the polypeptides in accordance with the invention, and also recombinant vectors comprising said polynucleotides, and host cells transformed with said vectors.
A subject of the present invention is also a composition comprising one or more polypeptide(s) a) and one or more polypeptide(s) b), as defined above, and also a composition comprising one or more polynucleotide(s) encoding said polypeptide(s).
The polypeptides a) and b) defined above can be used in the context of various methods for detecting antibodies, which are known in themselves. By way of examples, mention will in particular be made of ELISA type methods (direct, indirect or sandwich), methods of microagglutination on beads, and also methods of electrophoretic blotting coupled to immunolabeling.
A subject of the present invention is also a kit for detecting the presence of anti-Trichinella antibodies in a biological sample, characterized in that it comprises one or more polypeptide(s) a) and/or one or more polypeptide(s) b) as defined above, and, where appropriate, buffers and reagents suitable for constituting a reaction medium which allows the formation of an antigen/antibody complex, and, optionally, means for detecting said antigen/antibody complex.
Advantageously, said kit comprises a polypeptide a) and/or a polypeptide b) as defined above, immobilized on a solid support. By way of nonlimiting examples of solid supports that can be used, mention will be made of microtitration plates, beads, microbeads or microparticles, strips, etc.
Said kit may also comprise reference samples, such as one or more negative serum or sera and one or more positive serum or sera.
A subject of the present invention is also the use of a polypeptide a) or of a polypeptide b), as defined above, for preparing antibodies specifically directed against said polypeptide.
These polypeptides may be used in the context of various methods, known in themselves, for preparing antibodies. They may, for example (optionally after the addition of a suitable adjuvant), be used for the immunization of an animal. They may also be grafted onto an affinity chromatography support, in order to make it possible to purify the antibodies specifically directed against the polypeptide concerned, from a biological fluid. The biological fluid may, for example, be the serum of an animal immunized beforehand with the polypeptide concerned, or a hybridoma supernatant; it may also be the serum of an animal infested with Trichinella, from which it is desired to isolate a subpopulation of antibodies specifically directed against the polypeptide concerned.
The present invention also encompasses any antibodies specifically directed against a polypeptide a) or a polypeptide b) as defined above. They may be polyclonal or monoclonal antibodies. Preferred antibodies are those recognizing the PSSGSRPTYP epitope (SEQ ID NO: 5).
Antibodies specifically directed against a polypeptide can be obtained by various techniques known in themselves, and in particular by conventional methods comprising the immunization of an animal with the polypeptide concerned (with a suitable adjuvant optionally added thereto), and the recovery of its serum (for the production of polyclonal antibodies), or of its lymphocyte cells (for the production of monoclonal antibodies).
The polypeptides a) and b) defined above, and also the polynucleotides encoding these polypeptides, can be used for the preparation of immunogenic compositions, and in particular of anti-Trichinella vaccines.
A subject of the present invention is also an immunogenic composition comprising one or more polypeptide(s) a) and/or one or more polypeptide(s) b) as defined above, or one or more polynucleotide(s) encoding said polypeptide(s), combined with one or more adjuvant(s) for enhancing the immune response.
According to a preferred embodiment of an immunogenic composition in accordance with the invention, it is a vaccine.
A large variety of adjuvants for increasing the immunogenicity of peptides are known in themselves to those skilled in the art: by way of examples of adjuvants, mention will be made of alum (aluminum hydroxide), complete Freund's adjuvant or incomplete Freund's adjuvant (IFA), liposomes, and also virosomes (reconstituted viral envelopes), peptide derivatives of muramic acid, etc. In the case of a vaccine, a pharmacologically acceptable adjuvant will of course be chosen; by way of examples of preferred adjuvants, mention will be made of adjuvants of “water-in-oil” emulsion type, for example the adjuvants sold by the company SEPPIC under the names MONTANIDE ISA 70 and MONTANIDE ISA 775, and which are also described in patents EP 480 982, EP 825 875, U.S. Pat. No. 5,422,109, U.S. Pat. No. 6,251,407 and U.S. Pat. No. 6,610,309.
Where appropriate, in particular in the case of short peptides (≦30 amino acids), said polypeptide(s) may be coupled to a carrier protein.
By way of examples of carrier proteins, mention will in particular be made of KLH (keyhole limpet hemocyanin), bovine serum albumin (BSA), ovalbumin, tetanus toxoid or diphtheria toxoid. It is also possible to form a multiepitope composition, by associating several copies of the same peptide with one another, and optionally with other peptide epitopes, in the form of chimeric polypeptides, or by means of a polymeric chain, for example a polylysine.
If a polynucleotide is used as immunogene, the immunogenic composition may be in the form of a recombinant vector into which the polynucleotide(s) to be administered is (are) inserted. Use may, for example, be made of viral vectors such as poxviruses, andenoviruses, retroviruses, lentiviruses, herpesviruses and AAVs (adeno-associated viruses), etc. It may also be in the form of a nonpathogenic bacterium, transformed with one or more expression vectors containing said polynucleotide(s). The polynucleotide(s) may also be administered directly, in the form of naked DNA, or it (they) may be incorporated into liposomes. In the case of a vaccine, use will preferably be made of a nonpathogenic bacterium (for example a lactobacillus, or a nonpathogenic strain of Escherichia coli or of Salmonella suis), or a vector derived from a vaccinal viral strain; for example, a vector derived from a vaccinal strain of the pseudorabies (Aujeszky's disease) virus.
The present invention will be understood more clearly with the aid of the further description which follows, which refers to examples illustrating the use of the NBL1 and 411 antigens, for the early immunodiagnosis of trichinellosis.